Bacterial meningitis due to Escherichia coli K1 is a serious illness of the central nervous system. It is the most common pathogen causing meningitis in premature infants (46%). Mortality rates range from 5-30% in infected neonates and likely to increase drastically as the incidence of antibiotic resistant E. coli infections has been on the rise. Alternative strategies are therefore, urgently needed to prevent this deadly disease. A major gap in our understanding of the pathogenesis of meningitis is how E. coli finds a safe haven to survive and multiply in the host. We have discovered that although E. coli is phagocytosed by macrophages (M$), it is not killed but can survive and proliferate inside them. Survival of E. coli within M$ occurs when the E. coli outer membrane protein A (OmpA) binds to the Fc-gRI alpha chain (CD64) on the macrophage surface. This model is supported by our findings that 1) E. coli strains that either lack OmpA or express non-functional OmpA do not bind to CD64 and are killed by M$, 2) OmpA-mutant strains are incapable of inducing meningitis in the newborn mouse model, and 3) CD64-/- mice are resistant to E. coli induced meningitis. We have also found that gp96, which is expressed on the macrophage cell surface upon infection, is an essential co-factor for CD64-mediated phagocytosis and survival of E. coli in M$. Therefore, our hypothesis is that E. coli manipulates the macrophage function via OmpA interaction with CD64 and gp96 during the initial stages to evade immune responses.
The specific aims are, Aim 1: Examine why phagocytosis mediated by binding to CD64 and gp96 enables E. coli to survive inside M$. We will evaluate how the binding of E. coli to both CD64 and gp96 contributes to the production of nitric oxide by iNOS and responsible for modulating the signaling events of M$ for the survival.
Aim 2 tests whether OmpA interacts with distinct extracellular domains of CD64 to modulate the macrophage phagocytic activity by using site-directed mutagenesis of CD64. The binding pocket on CD64 interacting with OmpA will be further defined by identifying and using small molecules that prevent the binding. We further demonstrate that CD64 interacts with Filamin A and PKC-a in response to infection with E. coli. Therefore, Aim 3 will test the hypothesis that if the interaction of Filamin A and PKC-a with CD64 is critical for maneuvering the macrophage function by E. coli. The proposed research is innovative because, to date, direct interaction of any bacterial protein to CD64 has not been implicated in bacterial infections. Moreover, OmpA binding to CD64 to subvert the phagocytosis of the bacteria represent a novel phenomenon. The information from these studies will have a great impact on our understanding of the pathogenesis of E. coli meningitis and will also be useful for developing therapeutic approaches to treat and prevent meningitis.

Public Health Relevance

Neonatal meningitis due to E. coli K1 is a serious illness of the central nervous system with unacceptably high rates of morbidity and mortality throughout the world. Incomplete understanding of the steps involved in the pathogenesis has contributed to our inefficiency in developing new therapeutic strategies. Our studies demonstrated that E. coli utilizes a host immune system protein to divert its function and therefore, understanding the interaction between the bacteria and the host protein possibly suggests new methods to treat or prevent meningitis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS073115-03
Application #
8269940
Study Section
Immunity and Host Defense Study Section (IHD)
Program Officer
Wong, May
Project Start
2010-06-15
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$317,275
Indirect Cost
$102,900
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
052277936
City
Los Angeles
State
CA
Country
United States
Zip Code
90027
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